Methods and genotyping panels for detecting alleles, genomes, and transcriptomes

ABSTRACT

Disclosed are methods and genotyping panels for detecting alleles, genomes, and transcriptomes in admixtures of two individuals.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/645,129, filed on Dec. 22, 2009, which claims priority to the U.S.Provisional Patent Application Ser. No. 61/140,063, filed on Dec. 22,2008, U.S. Provisional Patent Application Ser. No. 61/166,188, filed onApr. 2, 2009, and U.S. Provisional Patent Application Ser. No.61/231,232, filed on Aug. 4, 2009, which are hereby incorporated byreference herein in their entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention is in the field of methods and genotyping panelsfor detecting alleles, genomes, and transcriptomes in admixtures of twoindividuals.

Description of the Related Art

Prenatal diagnostic methods are primarily aimed at obtaining geneticinformation of a fetus or an embryo. Prenatal genetic diagnostic methodsused in clinical practice essentially involve invasive techniques suchas amniocentesis, the removal of chorionic villi, and the removal offetal blood or tissue biopsies. Those techniques involve obtainingsamples directly from the fetus or indirectly from female reproductivestructures. Because of the highly invasive nature of those methods, theyare prone to complications for the mother or the fetus. Examples of suchcomplications which can be cited in the case of amniocentesis are therisk of infection, feto-maternal hemorrhage with possibleallo-immunization, loss of amniotic fluid and abdominal pain. Differentstudies have estimated the risk of a miscarriage after amniocentesis at0.06% to 2.1% higher than that of the control group (Eddleman, ObstetGynecol 2006 108(5): 1067-1072). As a result, amniocentesis is onlysuggested for women for whom the risk of having a child with aclinically significant genetic variation exceeds that of iatrogenicmiscarriage, and many physicians prefer to cite a risk commensurate withtheir experience (typically 1 in 300 to 1 in 500).

In order to limit the use of invasive prenatal diagnostic techniquesrisking the complications mentioned above and which are generallydisagreeable and/or the source of stress for the mother, the developmentof non-invasive methods constitutes a major aim in modern obstetrics.

In particular, fetal cells circulating in maternal blood constitute asource of genetic material that is of potential use for prenatal geneticdiagnosis (Bianchi, Br J Haematol 1999 105: 574-583; Fisk, Curr OpinObstet Gynecol 1998 10: 81-83). During pregnancy, different cell typesof fetal origin traverse the placenta and circulate in the maternalblood (Bianchi, Br J Haematol 1999 105: 574-583). Such cell typesinclude lymphoid and erythroid cells, myeloid precursors andtrophoblastic epithelial cells (cytotrophoblasts andsyncytiotrophoblasts).

Methods for analyzing the genome of fetal cells circulating in maternalblood with a view to prenatal diagnosis have been described, but theyremain relatively limited regarding sensitivity and the specificity ofthe diagnosis (Di Naro et al., Mol Hum Reprod 2000 6: 571-574; Watanabeet al., Hum Genet 1998 102: 611-615; Takabayashi et al., Prenat Diagn1995 15: 74-77; Sekizawa et al., Hum Genet 1998 102: 393-396). Theadvantage in developing a non-invasive, highly specific prenataldiagnosis method results from the possibility of using it to reduce theproportion of invasive diagnostic methods carried out in pregnant womenfor whom the result is negative in the end. By way of example, in thecase of trisomy 21, which concerns one woman in 700, prenatal diagnosisis currently offered in France only if the mother is 38 years old, whilea biochemical analytical test capable of detecting 60% of trisomy 21cases for 5% of the price of amniocentesis is proposed for youngerwomen. However, 40% of trisomy 21 cases are not detected by currentlyavailable tests. Prenatal detection of trisomy 21 in fetal cellsisolated from the maternal plasma using a FISH technique has beendescribed. That approach is interesting, but as fetal cells are rare inplasma (1 in 500 to 1 in 2000) and often include apoptotic cells,reliable diagnosis would require carrying out the method on a very largenumber of cells, rendering it impossible to carry out routinely.Further, euploid fetal cells cannot be identified by that approach.

One limitation of such approaches derives from the fact that fetal cellscirculating in the blood are present in very low concentrations. Studiesbased on PCR detection of the Y chromosome in blood samples withoutprior selection have allowed the mean number of fetal cells to bedetermined to be about one fetal lymphocyte cell per milliliter of blood(Bianchi, J Perinat Med 1998 26: 175-85). More recently, the mean numberof fetal cells has been revised upward as improved enrichment techniquesyield more cells. One recent study found a mean value of 37 fetallymphocytes per milliliter of blood. (Huang, Prenatal Diagnosis 2008 28:892-899).

Thus, there is a need for improved methods and tools for detecting fetalalleles and fetal genetic variations.

SUMMARY OF THE INVENTION

Embodiments of the present invention relate to methods and genotypingpanels for detecting fetal alleles and fetal genetic variations. Oneembodiment of the invention is a method of detecting a fetal identifier,comprising: obtaining a sample comprising a mixture of maternal andfetal cells; dividing the sample into subsamples; screening orgenotyping a subsample for the presence of a fetal identifier; andidentifying the presence of at least one fetal identifier from a fetalcell in at least one subsample.

Embodiments of the invention can and may comprise one or more of thefollowing: a method further comprising performing subsampleamplification on at least one subsample to provide an amplified product;a method further comprising dividing the amplified product intoaliquots, where screening or genotyping a subsample for the presence ofat least one fetal identifier comprises screening an aliquot for thepresence of a fetal identifier; a method where subsample amplificationcomprises a method selected from the group consisting of: whole genomeamplification, whole transcriptome amplification, targeted nucleic acidamplification, amplification of a nucleic acid sequence other than thefetal identifier, generation of a proxy for a nucleic acid sequence, andcell division; a method where preamplification comprises whole genomeamplification or whole transcriptome amplification; a method wherepreamplification comprises targeted nucleic acid amplification,amplification of a nucleic acid sequence other than the fetalidentifier, generation of a proxy for a nucleic acid sequence, and celldivision; a method further comprising determining whether the geneticmaterial of the maternal cells is homozygous or heterozygous at a set oftarget loci to determine a maternal genotype, where screening orgenotyping an aliquot or subsample for a fetal identifier comprisesscreening or genotyping the aliquot or subsample for a genotypediffering from the maternal genotype at at least one target locus, wherethe presence of the genotype differing from the maternal genotypeindicates the presence of an informative paternal allele in theamplified product, and where identifying the presence of at least onefetal identifier comprises identifying an informative paternal allele inat least one aliquot or subsample; a method further comprising selectingthe target loci prior to determining whether the genetic material of thematernal cells is homozygous or heterozygous; a method furthercomprising selecting a test locus from the target loci, where screeningcomprises screening an aliquot for a genotype differing from thematernal genotype at the test locus; a method where selection of thetest locus comprises screening a sample of mixed maternal and fetalnucleic acids for a genotype differing from the maternal genotype at atarget locus, and selecting as the test locus a target locus with anon-maternal genotype in the sample of mixed maternal and fetal nucleicacids; a method further comprising analyzing an aliquot or subsampleidentified as containing the informative paternal allele to detect agenetic variation; a method further comprising collecting a portion ofthe aliquot or subsample identified as containing the informativepaternal allele, where analyzing an aliquot or subsample identified ascontaining the informative paternal allele to detect a genetic variationcomprises analyzing the collected portion of the aliquot or subsample; amethod where the collected portion is a homogeneous portion of thealiquot or subsample; a method where the collected portion is anon-homogeneous portion of the aliquot; a method further comprisingcollecting an aliquot identified as containing the informative paternalallele from at least two subsamples and combining the aliquots prior toanalyzing the amplified product; a method where the genetic variation isa fetal genetic variation selected from the group consisting of achromosomal rearrangement, a copy number variation, and a polymorphism;a method where analysis comprises determining a ratio of maternally- andpaternally-inherited alleles in an aliquot comprising the informativepaternal allele, where the ratio is analyzed to determine the presenceof a genetic variation; a method where analysis comprises determining acopy number of alleles in an aliquot comprising the informative paternalallele, where the copy number of alleles is analyzed to determine thepresence of a genetic variation; a method further comprising analyzingan amplified product identified as containing the informative paternalallele from at least two the subsamples to detect the presence ofmosaicism or dizygotic twins; a method where the analyzed aliquot orsubsample is not the aliquot or subsample screened or genotyped for thepresence of the fetal identifier, but is from the same subsample as thealiquot screened or genotyped for the presence of the fetal identifier;a method where the target loci are all homozygous for the maternalgenotype, are all heterozygous for the maternal genotype, or comprise amixture of homozygous and heterozygous loci for the maternal genotype; amethod where the target loci are all homozygous for the maternalgenotype; a method where the amplified product comprises genomic DNA orcomplementary DNA; a method where determining a maternal genotypecomprises using a panel of SNPs to genotype a sample of maternal geneticmaterial from the same individual that is the source of the maternalcells; a method where the source of the maternal genetic material isselected from the group consisting of blood, serum, plasma, urine, acervical swab, tears, saliva, buccal swab, or skin; a method where thesource of the maternal genetic material is selected from the groupconsisting of blood or a buccal swab; a method where the mixture ofmaternal and fetal nucleic acids is a mixture of cell-free nucleicacids; a method where the source of the maternal and fetal cell-freenucleic acid sample is blood, serum, plasma, urine, cervical swab,cervical lavage, uterine lavage, or culdocentesis from a pregnantfemale; a method comprising selecting and screening or genotyping morethan one test locus in a single aliquot or subsample, selecting andscreening or genotyping more than one test locus in more than onealiquot or subsample, or selecting and screening or genotyping one testlocus in more than one aliquot or subsample; a method comprisingselecting and screening or genotyping a second target locus in at leasta second subsample following the identification of an informativepaternal allele in a first aliquot or subsample; a method where thesamples are divided to generate subsamples with a Poisson or aNon-Poisson distribution of cells; a method where each subsamplecomprises not more than one cell; a method further comprising poolingaliquots from two or more subsamples prior to screening or genotyping; amethod where pooling comprises the use of an indexed system of rows andcolumns of wells comprising the aliquots; a method further comprisingenriching the sample for the fetal cells prior to dividing intosubsamples; a method where enriching comprises differential expansion ofthe fetal cells over the maternal cells; a method where a homogeneousportion of the amplified product is divided into the aliquots; and amethod where a non-homogeneous portion of the amplified product isdivided into the aliquots.

Yet another embodiment of the invention is a single nucleotidepolymorphism (SNP) panel for detecting a fetal allele, comprising atleast one chromosome-specific panel comprising about 5 to about 100unique SNPs specific for a single chromosome, where each of the SNPs hasa frequency in the range of about 30% to about 50% as measured acrossall major population groups; and where the total number of SNPs in theSNP panel is between about 5 and about 100 SNPs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Embodiments of the present invention relate to methods and genotypingpanels for detecting fetal alleles (i.e., non-maternalalleles/informative paternal alleles) and fetal genetic variations.

Embodiments of the present invention allow for detection of fetalalleles and fetal genetic variations regardless of the gender of thefetus and without requiring a paternal nucleic acid sequence to serve asa reference sample. By distinguishing fetal cells from maternal cells,preferably on a cell-by-cell basis, embodiments of the inventionovercome resource constraints imposed by the low abundance of fetalcells in maternal blood and preserve the integrity of the fetal genomeor transcriptome for more extensive analysis than currently availablemethods. In addition, by distinguishing fetal cells on a cell-by-cellbasis, embodiments of the invention allow for the detection of mosaicismand dizygotic twins.

In a preferred embodiment of the invention, a maternal sample isgenotyped to identify homozygous target loci that are subsequentlygenotyped to detect the presence of fetal heterozygous loci in a samplecomprising fetal cells. It will be understood by one of skill in the artin any of the embodiments described herein that a maternal sample canalternatively be genotyped to identify heterozygous target loci that aresubsequently genotyped to detect the presence of fetal homozygous lociin a sample comprising fetal cells. In some embodiments, a mixture ofmaternal and fetal cells is obtained and enriched to generate a sampleconcentrated for fetal cells relative to maternal cells. Theconcentrated sample is then divided into subsamples such that eachsubsample preferably comprises only one cell. Each of these subsamplesis then amplified to produce an amplified product corresponding to eachsubsample, providing access to the genomes corresponding to cells.Amplified products are then divided to generate aliquots. These aliquotsare then individually screened for a non-maternal allele at at least onelocus that was previously identified as homozygous in the maternalsample or genotyped at at least one locus that was previously identifiedas heterozygous in the maternal sample, with detection of a heterozygousor homozygous genotype, respectively, indicating the presence of anon-maternal allele in the aliquot, and thus a fetal genome ortranscriptome. Detecting a heterozygous genotype may be accomplished byscreening for the non-maternal allele only at a selected locus. Furtheranalysis is then performed on the corresponding unscreened aliquots todetect a fetal genetic variation, with at least one aliquot comprising acomplete fetal genome or transcriptome.

In another preferred embodiment of the invention, a maternal sample isgenotyped, a mixture of maternal and fetal cells is obtained, and thesample is concentrated for fetal cells and divided into subsamples asdiscussed above. A panel of at least one target locus at which thematernal sample is homozygous is selected for screening or genotyping ofthe subsamples. Each of the subsamples is individually screened orgenotyped at at least one of these loci, with detection of aheterozygous genotype again indicating the presence of a non-maternalallele in the subsample. Analysis of the ratio of alleles in theheterozygous subsamples is then performed to detect a fetal copy numbervariation.

As used herein, nucleic acid means a deoxyribonucleic acid (e.g., DNA,mtDNA, gDNA, or cDNA), ribonucleic acid (e.g., RNA or mRNA), or anyother variant of nucleic acids known in the art.

As used herein, target locus means a genomic or transcriptomic locus atwhich a sample containing maternal genetic material has a detectablegenotype. In some embodiments with more than one target locus, thetarget loci are comprised of all homozygous loci, all heterozygous loci,or a mixture of homozygous and heterozygous loci. The panel of targetloci can also comprise all homozygous loci, all heterozygous loci, or amixture of homozygous and heterozygous loci.

As used herein, test locus means a target locus selected for furthergenotyping in a sample comprising fetal genetic material.

As used herein, a fetal identifier refers to any indicator that a sampleor portion thereof is fetal in origin. A fetal identifier can includeany genetic variation or other information. One example of a fetalidentifier is a fetal allele. As used herein, a fetal allele refers to apaternal allele that provides the ability to identify a genetic sourceas fetal. A paternal allele is any allele present in a paternal sample.However, as used herein, an informative paternal allele is any alleleequivalent to a non-maternal allele (as used herein) or a fetal allele(as used herein).

As used herein, genetic variation means any variation in a nucleic acidsequence. Genetic variations can range from a single base pair variationto a chromosomal variation, or any other variation known in the art.Genetic variations can be simple sequence repeats, short tandem repeats,single nucleotide polymorphisms, translocations, inversions, deletions,duplications, or any other copy number variations. In some embodiments,the chromosomal variation is a chromosomal abnormality. For example, thechromosomal variation can be aneuploidy, inversion, translocation, adeletion, or a duplication. A genetic variation can also be mosaic. Forexample, the genetic variation can be associated with genetic conditionsor risk factors for genetic conditions (e.g., cystic fibrosis, Tay-Sachsdisease, Huntington disease, Alzheimer disease, and various cancers).Genetic variations can also include any mutation, chromosomalabnormality, or other variation disclosed in the priority documents(e.g., aneuploidy, microdeletions, or microduplications) cited above.Genetic variations can have positive, negative, or neutral effects onphenotype. For example, chromosomal variations can include advantageous,deleterious, or neutral variations. In some embodiments, the geneticvariation is a risk factor for a disease or disorder. In someembodiments, the genetic variation encodes a desired phenotypic trait.

Obtain a Sample (e.g., Comprising a Mixture of Maternal and Fetal Cells)

The maternal samples, samples of mixed maternal and fetal cells, andsamples of mixed maternal and fetal cell-free nucleic acids inembodiments of the invention can be obtained from blood. In someembodiments, about 20-40 mL of blood is drawn from a pregnant woman.Blood samples can be collected at any point during pregnancy. Forexample, in some embodiments, the maternal sample is collected duringthe first trimester. In other embodiments, the maternal sample iscollected during the second trimester. In a preferred embodiment, bloodis drawn at 10-18 weeks gestational age. However, blood can be drawnearlier in the pregnancy or after 18 weeks gestational age. The time ofcollection may vary depending on the information sought or the standardsof prenatal care. Blood samples can also be collected at any time duringthe day. In some embodiments, blood is collected in the morning. Inother embodiments, blood is collected in the afternoon.

Blood can be drawn from any suitable area of the body, including an arm,a leg, or blood accessible through a central venous catheter. In someembodiments, blood is collected following a treatment or activity. Forexample, blood can be collected following a pelvic exam. The timing ofcollection can also be coordinated to increase the number of fetal cellspresent in the sample. For example, blood can be collected followingexercise or a treatment that induces vascular dilation.

Blood may be combined with various components following collection topreserve or prepare samples for subsequent techniques. For example, insome embodiments, blood is treated with an anticoagulant, a cellfixative, or a DNA or RNA preservative following collection. In apreferred embodiment, blood is collected via venipuncture using vacuumcollection tubes containing an anticoagulant such as EDTA or heparin.Blood can also be collected using a heparin-coated syringe andhypodermic needle. Blood can also be combined with components that willbe useful for cell culture. For example, in some embodiments, blood iscombined with cell culture media or supplemented cell culture media(e.g., cytokines).

Maternal samples can also be obtained from other sources known in theart, including serum, plasma, urine, cervical swab, tears, saliva,buccal swab, skin, or other tissues. Samples of mixed maternal and fetalcells and samples of mixed maternal and fetal cell-free nucleic acidscan also be obtained from other sources known in the art, includingserum, plasma, urine, cervical swab, cervical lavage, uterine lavage,culdocentesis, lymph node, or bone marrow. For example, in someembodiments, the source of a sample of mixed maternal and fetalcell-free nucleic acids is a cervical swab. In a preferred embodiment,the fetal cell-free nucleic acids comprise DNA. In another embodiment,the fetal cell-free nucleic acids comprise RNA or cDNA.

Enrich for Fetal Cells

In order to address the low abundance of fetal cells in mixed samples ofmaternal and fetal cells, these samples can be enriched for fetal cells.Because red blood cells are enucleated when mature, but nucleated whenimmature, these properties can be used to differentiate maternal andfetal red blood cells in a sample. Samples can be enriched for fetalcells through positive selection, negative selection, or a combinationof positive and negative selection. In some embodiments, fetal cells aredirectly captured. In other embodiments, maternal cells are captured andfetal cells are collected from the remaining sample.

Samples can be enriched for fetal cells based on differences in thephysical properties of cells. For example, samples can be enriched forfetal cells based on density, cell membrane structure, or morphology. Insome of the embodiments based on density, density gradients such asFICOLL™ (GE Healthcare Life Sciences, Piscataway, N.J.), PERCOLL™ (GEHealthcare Life Sciences, Piscataway, N.J.), iodixanol (Axis Shield,Oslo, Norway), NYCODENZ® (Axis Shield, Oslo, Norway), or sucrose areused. In some of the embodiments based on cell membrane structure, alysis reagent (e.g., ammonium chloride) is used. In some of theembodiments based on morphology, flow cytometry or filters are used.Samples can also be enriched for fetal cells based on other physicalproperties known in the art. For example, samples can be enriched forfetal cells based on dielectric or magnetic properties. Further, samplescan be enriched for fetal cells by collecting bone marrow.

Samples can also be enriched for fetal cells based on differences in thebiochemical properties of cells. For example, samples can be enrichedfor fetal cells based on antigen, nucleic acid, metabolic, geneexpression, or epigenetic differences. In some of the embodiments basedon antigen differences, antibody-conjugated magnetic or paramagneticbeads in magnetic field gradients or fluorescently labeled antibodieswith flow cytometry are used. In some of the embodiments based onnucleic acid differences, flow cytometry is used. In some of theembodiments based on metabolic differences, dye uptake/exclusionmeasured by flow cytometry or another sorting technology is used. Insome of the embodiments based on gene expression, cell culture withcytokines is used. In some of the embodiments based on epigeneticdifferences, cell culture is used. Samples can also be enriched forfetal cells based on other biochemical properties known in the art. Forexample, samples can be enriched for fetal cells based on pH ormotility. Further, in some embodiments, more than one method is used toenrich for fetal cells.

In some embodiments of the invention, samples are enriched for fetalcells by removing red blood cells through the use of lysis reagents suchas ammonium chloride or by separation using density gradients such asFICOLL™ (Sigma-Aldrich, St. Louis, Mo.), PERCOLL™ (GE Healthcare LifeSciences Piscataway, N.J.), or sucrose. A density gradient can also beused to reduce the white cell fraction. The resulting peripheral bloodmononuclear cells (“PBMCs”) can be further enriched for fetal cellsusing magnetic bead separation techniques from manufactures such asMiltenyi Biotec (Gladbach, Germany), Stemcell Technologies (Vancouver,BC, Canada), and Dynal Biotech/Invitrogen (Carlsbad, Calif.). Positiveenrichment or negative depletion or a combination of both can be used toenrich the fetal fraction in the PBMCs.

While no fetal specific surface markers are currently known, there areseveral markers that have been shown to positively enrich fetal cells to1 fetal cell in 1,000 to 100,000 maternal cells. In some embodiments,CD71, CD34, CD45, or CD235a cell surface markers are used to enrichfetal cells. In some embodiments, cell surface markers that are notfound on fetal cell populations are used to negatively enrich fetalcells by depleting adult cell populations. In some embodiments,combinations of CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123 andCD61 are used to deplete adult cells. Flow cytometry sorting may also beused to further enrich for fetal cells using cell surface markers orintracellular markers conjugated to fluorescent labels. Intracellularmarkers may include nuclear stains or antibodies against intracellularproteins preferentially expressed in fetal cells (e.g., fetalhemoglobin).

Oxidation of hemoglobin has been identified as one way to preferentiallyenrich nucleated red blood cells (NRBCs) using magnetic field gradients(Zborowski, Biophys J 2003 84(4): 2638-2645). In addition, microfluidicdevices have been developed which facilitate separation of red cellsfrom white cells or enrich fetal cells from PBMCs (Huang, PrenatalDiagnosis 2008 28: 892-899).

In some embodiments of the invention, samples are enriched for fetalcells by differentially expanding fetal cells over maternal cells inculture. Differential expansion can be performed by any number ofmethods known in the art, including incubating cells from a sample ofmaternal blood containing CD34+ cells of both maternal and fetal originin the presence of Stem Cell Factor (SCF) in serum free media asdescribed in WO 2008/048931, which is herein incorporated by referencein its entirety. In some embodiments of the invention, fetal cells in amixture of maternal and fetal cells are enriched to about 1 in 2, about1 in 5, about 1 in 10, about 1 in 100, about 1 in 1000, about 1 in10000, or about 1 in 100000 fetal:maternal cells, or a range defined byany two of the preceding values.

Divide into Subsamples

Prior to subsample amplification (preferably using whole genomeamplification (WGA) or whole transcriptome amplification (WTA)),screening or genotyping for homozygous or heterozygous loci, the samplescan be divided into subsamples with few enough cells such that thechromosome copy number from the samples is preserved in the subsampleseven following subsample amplification (e.g., WGA or WTA). Samples canbe divided into subsamples consistent with a Poisson Distribution or aNon-Poisson Distribution. In some embodiments, samples are dividedsequentially. For example, samples can be divided in serial. In otherembodiments, samples are divided in parallel.

In some embodiments, samples are divided to provide subsample volumesof, less than, or less than about, 100 uL, 50 uL, 10 uL, 1000 nL, 500nL, 400 nL, 300 nL, 200 nL, 100 nL, 50 nL, 30 nL, 10 nL, 3 nL, or 1 nL,or a range defined by any two of the preceding values. Preferably, eachsubsample contains a volume not more than 100 nL. In some embodiments,each subsample comprises not more than about 500, 400, 300, 200, or 100cells, or a range defined by any two of the preceding values.Preferably, each subsample comprises not more than about 50, 40, 30, 20,or 10 cells, or a range defined by any two of the preceding values. Morepreferably, each subsample comprises not more than about 5, 4, 3, or 2cells. In some embodiments, each subsample comprises not more than onecell.

In some embodiments, each subsample comprises an average of, or ofabout, 500, 400, 300, 200, or 100 cells, or a range defined by any twoof the preceding values. Preferably, each subsample comprises an averageof about 50, 40, 30, 20, or 10 cells, or a range defined by any two ofthe preceding values. More preferably, each subsample comprises anaverage of, or of about, 5, 4, 3, or 2 cells, or a range defined by anytwo of the preceding values. In some embodiments, each subsamplecomprises an average of less than about one cell, about one cell, orabout one to two cells, or a range defined by any two of the precedingvalues.

The division of samples is performed by any method known in the art,including the use of oil plugs to create oil separation of individualcells in a microfluidic device, deposition into wells, or free-standingdrops anchored by surface tension to a flat substrate. In addition, asubsample can be suspended in a buffer that will be appropriate forsubsequent reactions. For example, a subsample can suspended in asolution comprising lysis and PCR buffers that will allow for asingle-step cell lysis followed by amplification without furthermanipulation of subsamples.

Amplify Subsamples

To compensate for the limited amount of genetic material in a singlecell or subsample, subsample amplification can optionally be performed.For example, nucleic acid replication or cell division can be performed.Samples are divided into subsamples with few enough cells such that thechromosome copy number from the samples is preserved in the subsamplesfollowing subsample amplification. In a preferred embodiment, subsampleamplification is performed on a subsample containing a single cell, sothat the resulting amplified product represents the genome ortranscriptome of either a maternal or fetal cell. For example, subsampleamplification can be performed on individual cells that are located inmicrowells or in drops separated by oil plugs as described herein.

Nucleic acid replication can be performed using any method forgenerating additional copies of nucleic acids, additional signalsindicative of nucleic acids, or other proxies for nucleic acids (e.g.,protein expression) known in the art. In some embodiments, nucleic acidreplication is performed using WGA, WTA, or targeted nucleic acidamplification techniques. In other embodiments, nucleic acid replicationis performed using methods that generate a signal indicative of nucleicacid sequences, such as INVADER® (Hologic, Inc., Bedford, Mass.). Insome embodiments, only a portion of the amplified sequence iscomplementary to the nucleic acid template. For example, in someembodiments, a contiguous amplified product contains a portion of thenucleic acid template and a portion of a signal sequence. Generaltechniques for nucleic acid replication can include isothermal orthermocycled replication. In some embodiments, nucleic acid replicationis performed prior to SNP genotyping. However, nucleic acid replicationcan also be performed after SNP genotyping.

Cell replication can also be performed using any method known in theart. In some embodiments, cells are cultured in media and supplements togenerate additional nucleic acid copies for use in the methods describedherein. In other embodiments, cells are cultured and one or more cell isleft intact for use in subsequent analysis. In some embodiments, cellreplication is performed prior to division into subsamples. Preferably,cell replication is performed after division into subsamples.

Divide into Aliquots

Following subsample amplification, amplified products can be dividedinto aliquots. These aliquots can be used for a plurality of assays. Forexample, in one embodiment, one or more of the aliquots from anamplified product is used to detect the presence of a fetal allele,while one or more of the other aliquots is used to detect the presenceof a fetal genetic variation in an amplified product that contains afetal genome or transcriptome. In some embodiments, an aliquotidentified as containing a fetal genome or transcriptome is assayed byarray for genetic variations. For example, an aliquot can be assayed forgenetic variations associated with genetic conditions (e.g., Williamssyndrome, Wolf-Hirschhorn syndrome, Miller-Dieker syndrome, SmithMagenis syndrome, Angelman syndrome, Di George syndrome, Prader-Willisyndrome, Jacobsen syndrome, Cri du chat syndrome, Charcot-Marie-Toothdisease, microduplication 22q11.2 syndrome, cystic fibrosis, Tay-Sachsdisease, Huntington disease, Alzheimer disease, and various cancers).

Homogenous or non-homogeneous portions of amplified products can beselected for division into aliquots. In some embodiments, homogenousportions of amplified products are divided sequentially (e.g., inserial). In other embodiments, homogeneous portions of amplifiedproducts are divided in parallel. Alternatively, non-homogeneousportions of amplified products can be selected for division intoaliquots using positive selection, negative selection, or a combinationof positive and negative selection. For example, in some embodiments,bead-bound capture oligos are used to target desired portions ofamplified products for division into aliquots. In other embodiments,surface-bound oligos are used to eliminate undesired portions ofamplified products. Non-homogeneous portions of amplified products canbe selected based on any physical or biochemical property known in theart, including those described herein. For example, portions ofamplified products with a particular charge, size, or chromosomalidentity can be selected for division into aliquots.

In some embodiments where the optional step of subsample amplificationis not carried out, a portion or aliquot of a subsample can be removedfor subsequent analysis.

In some embodiments, aliquots are pooled into groups of two or morealiquots. This allows the number of SNP-based or other reactionsdescribed herein to be reduced by as much as a factor of N, where N isthe number of aliquots in each pool. Aliquots from positive pools (i.e.,pools with at least one genotype differing from the maternal genotype)may then be retested aliquot-by-aliquot to identify the aliquotcontaining a fetal allele. In some embodiments, each pool is tested fora non-maternal allele at a test locus. In some embodiments, each pool istested for non-maternal alleles at two or more test loci.

In some embodiments, aliquots are pooled using an indexing system thatallows for identification of the source of a positive aliquot within apositive pool. For example, two or more aliquots may be taken from eachamplified product to form indexed pools of N×M amplified aliquots. Wellscontaining at least one fetal allele can be identified by locating theintersection of positive N and M pools in the orthogonal ordinatesystem. In some embodiments, N (i.e., the number of columns in the N×Mindex) and M (i.e., the number of rows in the N×M index) areindependently between about 2 and about 1000. Preferably, N and M areindependently between about 8 and about 100. In some embodiments, N is,is about, is at least, is at least about, is not more than, is not morethan about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 30, 32, 36, 40, 48, 50, 56, 60, 64, 70, 72, 80,84, 88, 90, 96, 100, 192, 288, 384, 480, 576, 672, 768, 864, 960, 1000,or a range defined by any two of the preceding values. In someembodiments, M is, is about, is at least, is at least about, is not morethan, is not more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 32, 36, 40, 48, 50, 56, 60,64, 70, 72, 80, 84, 88, 90, 96, 100, 192, 288, 384, 480, 576, 672, 768,864, 960, 1000, or a range defined by any two of the preceding values.In preferred embodiments, homogeneous or non-homogeneous portions ofamplified products are indexed to allow for identification of the sourceof positive aliquots.

Obtain or Infer a Parental Genotype

Parental genotypes can be obtained or inferred to aid in theidentification of non-maternal alleles. Information regardingnon-maternal alleles can in turn be used to screen or genotype aliquotsor subsamples, or to directly analyze fetal genomes for geneticvariants. For example, paternal or maternal genotypes can be obtained bydirectly genotyping paternal or maternal samples, or inferred bygenotyping samples from genetically related family members. However, ina preferred embodiment, there is no need to obtain or infer a paternalgenotype. For example, in a preferred embodiment, only a maternalgenotype is obtained.

In some embodiments, paternal or maternal genotypes are obtained bygenotyping genetic material from blood, plasma, serum, urine, buccalswab, saliva, tears, skin, or any other source of paternal nucleic acids(including those described herein). In some embodiments, paternal ormaternal genotypes are obtained using cell-free nucleic acids or nucleicacids extracted from cells derived from one of these sources. Paternalor maternal genotyping is also preferably performed on DNA, but can alsobe performed on RNA, cDNA, or any other nucleic acid known in the art.In some embodiments, the template of a paternal or maternal sample isamplified and detected (e.g., using PCR-based methods). However, in someembodiments, the template of a paternal or maternal sample is notamplified (e.g., using the methods described herein).

Paternal and maternal genotypes can also be obtained by accessinginformation generated during prior genetic testing, such as informationin a database, in a test report, or from a previous pregnancy for whicha method described herein was performed. Paternal or maternal genotypescan also be inferred using the genotypes of blood relatives. Forexample, the genotypes of genetically related parents, siblings,grandparents, aunts, uncles, or children can be used to infer a paternalor maternal genotype.

Any polymorphism known in the art can be used to genotype a parentalsample. For example, SNPs, haplotypes, short tandem repeats (STRs), orother sequence variations can be genotyped. Other genetic or epigeneticmarkers can also be used to genotype a parental sample. For example,copy number variations (CNVs) or methylation patterns can be assessed.

Optionally Identify at Least One Non-Maternal Allele in a Mixture ofMaternal and Fetal Nucleic Acids

In embodiments where a homozygous target locus has been identified in amaternal sample, a mixture of maternal and fetal nucleic acids canoptionally be used to identify a heterozygous genotype at the samelocus, which indicates the presence of a fetal (i.e., non-maternalallele/informative paternal allele). This optional step is preferablyperformed prior to screening or genotyping the individual aliquots orsubsamples. By screening for a non-maternal allele, and therebyidentifying a heterozygous locus (and therefore a fetal allele) in themixed sample of maternal and fetal nucleic acids, the aliquots andsubsamples can be more efficiently screened for SNPs that are known tobe informative. In a preferred embodiment, fetal cell-free DNA is usedto screen for the non-maternal allele and identify the heterozygouslocus. In another embodiment, fetal cell-free RNA or cDNA is used toidentify the heterozygous locus.

Cell-free nucleic acids can be obtained from any source known in theart, including blood, serum, plasma, urine, cervical swab, cervicallavage, uterine lavage, or culdocentesis from a pregnant woman. Nucleicacids can also be extracted from a mixed sample of maternal and fetalcells to identify the heterozygous locus. Preferably, DNA is extractedfrom a mixed sample of maternal and fetal cells. However, RNA or cDNAcan be extracted from a mixed sample of maternal and fetal cells.Nucleic acids can be extracted from cells obtained from any source knownin the art, including blood, cervical swab, cervical lavage, uterinelavage, culdocentesis, lymph node, or bone marrow. In other embodiments,whole blood is used to identify a heterozygous genotype without (orprior to) dividing the whole blood into a cellular, plasma, or serumfraction.

In some embodiments, the nucleic acid template of an aliquot from amixed sample of maternal and fetal nucleic acids is amplified anddetected to identify a heterozygous locus (e.g., using PCR-basedmethods). However, in some embodiments, the nucleic acid template of analiquot from a mixed sample is not amplified to identify a heterozygouslocus. For example, methods in which only a signal associated with thetemplate is amplified (e.g., the ABSCRIPTION™ method as described inU.S. Pat. Nos. 7,226,798, 7,473,775, and 7,468,261 (RibomedBiotechnologies, Inc., Carlsbad, Calif.) or methods involving theINVADER® chemistry (Hologic, Inc.)) can be employed. Methods in whichthe nucleic acid template is detected without amplification of thesignal or the template (e.g., the method involving chemical detection ofDNA binding as described in WO 2005/01122 (Adnavance Technologies, Inc.,San Diego, Calif.)) can also be employed. In addition, methods in whichthe nucleic acid template is sequenced without amplification (e.g., thesequencing method as described in Eid et al., Science 2009 323(5910):133-38) can be employed. It will be understood by one of skill in theart that the source of the template for the identification of anon-maternal allele can include serum, plasma, urine, a cervical swab,or any other source of nucleic acids known in the art.

Screen or Genotype at Least One Aliquot or Subsample for the Presence ofat Least One Informative Paternal Allele

The next steps are to screen or genotype subsamples or aliquots toidentify non-maternal alleles. Once a SNP panel has been generated (asdescribed in more detail herein) and a set of target loci for which thematernal genetic sample is homozygous (or, alternatively, heterozygous)has been identified, a test locus or loci can be selected to screen orgenotype the subsample or aliquot for the presence of a fetal allele. Todetect the presence of a fetal allele, a test locus is screened orgenotyped for the presence of a non-maternal allele (i.e., byidentifying a heterozygous, or alternatively homozygous genotype at thetest locus). In some embodiments, aliquots of an amplified product arescreened or genotyped aliquot-by-aliquot to detect a heterozygous (or,alternatively, homozygous) locus. In some embodiments, an aliquotcontains amplified material from a single cell.

A subsample or aliquot can be genotyped at a locus previously identifiedas homozygous in a maternal sample. In some embodiments, the genotype ata locus previously identified as homozygous in a maternal sample isdetermined by screening for the presence of a non-maternal allele.Optionally, as described herein, prior to screening the aliquots orsubsample for the non-maternal allele, a sample of mixed maternal andfetal nucleic acids (preferably cell-free) was used to identify thepresence of a non-maternal allele in the mixed nucleic acid, indicatingthe presence of a heterozygous genotype at the same locus in the fetalmaterial.

In other embodiments, a subsample or aliquot is genotyped at a locuspreviously identified as heterozygous in a maternal sample. Theidentification of a homozygous genotype in the aliquot or subsample forthe same locus indicates the presence of a non-maternal, i.e., fetalallele.

It will be understood by one of skill in the art that the screening orgenotyping methods in any of the embodiments described herein may beperformed using either aliquots or subsamples. Aliquots and subsamplescan be screened or genotyped for a fetal allele using a number ofmethods known in the art, including those mentioned herein. For example,aliquots can be screened or genotyped using molecular beacons or othernucleic acid-based SNP detection methods. In some embodiments, thenucleic acid template in an aliquot is amplified and detected (e.g.,using PCR-based methods). However, in some embodiments, the nucleic acidtemplate is not amplified (e.g., using the methods described herein).Further, any marker known in the art can be used to screen or genotypealiquots and subsamples. In preferred embodiments, the SNP, haplotype,short tandem repeat (STR), other sequence variation, copy numbervariation (CNV), or epigenetic marker genotyped in the maternal sampleis used.

Because in some embodiments subsamples comprise not more than one cell,the screening or genotyping of a subsample or aliquot can becell-by-cell. Subsamples or aliquots can also be screened or genotypedusing a number of methods known in the art, including those mentionedherein. Preferably, subsamples are screened for heterozygous allelesusing quantitative PCR (qPCR) with a TAQMAN® system (Foster City,Calif.). A predetermined number of subsamples or cells can be screenedto detect a heterozygous allele. For example, as shown in Table 1, totest 7 loci in a sample enriched to 1:10,000 fetal:maternal cells, whereapproximately 5 fetal cells per loci are expected, the predeterminednumber of samples or cells is 350,000. In some embodiments, the numberof fetal cells per loci is, is about, is at least, is at least about, isnot more than, is not more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225,250, 275, or 300, or a range defined by any two of the preceding values.

TABLE 1 Total Enriched Cells (in Thousands) 50 100 150 200 250 300 350400 Number of loci possible with 5 fetal cells per loci Fetal 1:100 100200 300 400 500 600 700 800 Fre- 1:1,000 10 20 30 40 50 60 70 80 quency1:10,000 1 2 3 4 5 6 7 8Identify at Least One Informative Paternal Allele in at Least oneAliquot or Subsample

As described herein, after homozygous (or, alternatively, heterozygous)SNPs are identified in maternal genetic material, these SNPs can bescreened or genotyped in the subsamples or aliquots to detect thepresence of a non-maternal allele. If a first maternalhomozygous/heterozygous SNP does not generate a heterozygous/homozygousgenotype in the aliquots or subsamples, a second maternalhomozygous/heterozygous SNP can be selected and genotyped. This processcan be repeated until a non-maternal allele is detected or until apredetermined number of SNPs and/or cells, subsamples or aliquots arescreened. The process can also involve genotyping multiple aliquots orsubsamples, multiplexing SNPs, or any combination thereof.

Test loci are screened or genotyped such that at least one fetal allelewill be detected if present in a sample. In some embodiments, test lociare screened or genotyped until at least one non-maternal allele isdetected. In some embodiments, a first test locus is screened orgenotyped in aliquots or subsamples until enough cells are genotyped toexceed a designated probability of detecting a non-maternal allele. If anon-maternal allele is not detected, a second test locus is screened orgenotyped in aliquots or subsamples until enough cells are run to exceeda designated probability of detecting a non-maternal allele. If anon-maternal allele is not detected, additional test loci are screenedor genotyped until the number of test loci run exceeds a designatedcumulative probability of detecting a non-maternal (and therefore fetal)allele from a mixed sample.

Test loci can also be screened or genotyped until more than one fetalallele is detected. If more than one test locus is screened orgenotyped, each additional locus testing positive for a fetal allele(i.e., with a genotype differing from the maternal genotype) increasesthe confidence of detecting a fetal genome in a subsample or aliquot. Inaddition, a predetermined number of test loci can be designated toaccount for the fact that fetal genetic material may not be present. Insome embodiments, a predetermined number of test loci is determined bycalculating the cumulative probability of detecting a fetal allele for arelevant set of variables. For example, as shown in Table 3, theprobability of detecting the presence of a heterozygous fetal alleleusing 7 SNPs with a minor allele frequency of about 0.5 is1−(0.5)(0.5)(0.5)(0.5)(0.5)(0.5)(0.5)=99.22%. As shown in Table 1, for350,000 aliquots or subsamples from a sample enriched for 1:10,000fetal:maternal cells, the predetermined number of test loci cantherefore be about 7 test loci.

Test loci can be screened or genotyped individually or in multiples. Insome embodiments, one test locus is screened or genotyped in a singlealiquot or subsample. A single test locus can also be screened orgenotyped in multiple aliquots or subsamples. In addition, aliquots orsubsamples can be screened or genotyped individually or in multiples. Insome embodiments, more than one test locus is screened or genotyped in asingle aliquot or subsample. In addition, more than one test locus canalso be screened or genotyped in multiple aliquots or subsamples. Insome embodiments, the number of test loci in a panel is, is about, is atleast, is at least about, is not more than, is not more than about 2, 3,4, 5, 6, 7, 8, 9, or 10 test loci multiplexed to screen for or genotypea fetal allele, or a range defined by any two of the preceding values.

In some embodiments, a locus identified as containing a heterozygous(and therefore non-maternal) genotype in a mixture of maternal and fetalnucleic acids (preferably cell-free) is screened subsample-by-subsamplewith subsamples derived from a mixture of maternal and fetal cells.Conserved aliquots and subsamples can then be used to perform additionalgenetic analyses. However, despite the advantages of maternal and fetalcell-free nucleic acids in detecting the presence of a fetal allele,these samples are not suitable for analyses that require preservation ofthe integrity of the fetal genome or transcriptome, or capture ofsamples. This highlights one of the benefits of using the cell-basedmethods described herein.

Collect Aliquots or Subsamples

Aliquots or subsamples identified as containing a fetal allele can becollected for subsequent analyses. The aliquot(s) collected forsubsequent analysis can be the same one(s) used to screen for the fetalallele, or a different aliquot from the same subsample can be used toprovide an aliquot that has not be subject to any reactions used forfetal allele screening. In some embodiments, aliquots or subsamples areselected for collection based on quality, quantity, or the presence ofdesired nucleic acids. For example, aliquots or subsamples can beselected for collection using signal correlations, signal intensities,signal intensity ratios, signals compared to a background measurement,assay kinetics plotted against time, assay kinetics plotted againsttemperature, or other performance metrics known in the art. In someembodiments, the marker or region used to discriminate paternal frommaternal alleles is collected. However, in other embodiments, anunlinked marker or region is collected for further analysis.

In some embodiments, only a desired portion of an aliquot or subsampleis collected for subsequent analysis. For example, in some embodiments,a hybridization probe is used to collect only nucleic acid sequencesfrom a chromosome or region of interest. In other embodiments, an entirealiquot, subsample, or a homogenous portion thereof is collected.

Analyze at Least One Fetal Genome for Genetic Variants

Aliquots or subsamples identified as having a non-maternal allele,optionally collected as described herein, can be further analyzed, forexample, to test for the presence of a chromosomal or genetic variation.The aliquot used for analysis can be the same one screened or genotypedfor the fetal allele. Or the aliquot used for analysis can be adifferent aliquot from the same subsample, but one that was not subjectto any screening or genotyping for a fetal allele. Entire fetal genomesor portions thereof can be selected for further analyses. In someembodiments, polymorphisms are genotyped using methods known in the art.For example, SNPs, haplotypes, or STRs can be genotyped using PCR and,if appropriate, subsequent detection methods such as capillaryelectrophoresis. Polymorphisms can also be genotyped using sequencingmethods. Genotyping is preferably performed using high throughputtechniques. For example, in some embodiments, a microarray is used togenerate data regarding SNPs and/or haplotypes. Copy number variationcan also be assessed for further analyses. For example, arraycomparative genomic hybridization (aCGH) can be used to detect copynumber variations. Chromosomal rearrangements can also be assessed. Forexample, inversions or translocations can be detected using methods suchas sequencing, FISH, or PCR.

In some embodiments, a ratio of maternally- and paternally-inheritedalleles is determined to analyze the presence of a genetic variation.Optionally, the same locus is used to determine the presence of a fetalallele and the presence of a genetic variation. For example, intensityof the alleles at a heterozygous test locus can be measured, with a 2:1or 1:2 intensity ratio indicating copy number variation. However, insome embodiments, the locus used to determine the presence of a fetalallele is not the same locus used to determine a genetic variation. Inaddition, it will be understood by one of skill in the art in any of theembodiments described herein that other intensity ratios (e.g., 3:1,1:3, 3:2, 2:3, 4:1, and 1:4) can be used to detect the presence of copynumber variation.

In some embodiments, an overrepresentation or underrepresentation ofchromosomal sequences is determined to analyze the presence of copynumber variation. For example, the number of unique sequence reads for aparticular chromosome can be measured and compared to a maternal and/orother reference chromosome, with a ratio less than or greater than 1:1indicating a copy number variation. The detection of these uniquesequence reads can be performed using small scale (e.g., sequencing withprimer pairs designed for specific loci) or large scale (e.g.,sequencing of the entire genome) methods. The number of sequence readsfor a particular chromosomal region can also be measured and compared toa maternal and/or other reference chromosomal region, with a ratio lessthan or greater than 1:1 indicating the presence of a copy numbervariation.

Aliquots or subsamples may be analyzed individually or in a combinedsample of at least two aliquots or subsamples. In some embodiments,aliquots or subsamples are ranked based on signal metrics as describedherein and a preferred set is selected for analysis or pooling followedby analysis. In some embodiments, isolated aliquots or subsamples orpools are tested for the presence of a genetic or chromosomal variationusing array comparative genomic hybridization (aCGH), quantitativefluorescence PCR (QF-PCR), short tandem repeat (STR) analysis, orsequencing. However, any technique known in the art, including thosedescribed herein, can be used to test for the presence of a genetic orchromosomal variation.

Screening or genotyping aliquots or subsamples on a cell-by-cell basisallows for the detection of mosaicism (i.e., a condition in which cellsfrom the same individual have different genetic profiles). For example,subsamples can be screened or genotyped to detect a mosaic chromosomalvariation. In some embodiments, the number of subsamples screened orgenotyped for mosaicism is about 2 to about 100 subsamples. In someembodiments, the number of subsamples screened or genotyped is, isabout, is at least, is at least about, is not more than, is not morethan about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60,70, 80, 90, or 100, or a range defined by any two of the precedingvalues. In a preferred embodiment, the number of subsamples screened orgenotyped is about 5 to about 10 subsamples. Optionally, the same locusis used to determine the presence of a fetal allele and the presence ofmosaicism. For example, intensity of the alleles at a heterozygous testlocus can be measured, with a 1:1 intensity ratio in at least onesubsample and a 2:1 or 1:2 intensity ratio in at least one othersubsample indicating the presence of a mosaic genetic variation.However, different loci can also be used to determine the presence of afetal allele and the presence of mosaicism. For example, a homozygoustest locus can be used to identify a fetal allele. A heterozygous locuscan then be detected and the intensity of the alleles at theheterozygous locus can be measured, with a 1:1 intensity ratio in atleast one subsample and a 2:1 or 1:2 intensity ratio in at least oneother subsample again indicating the presence of a mosaic geneticvariation.

In some embodiments, mosaicism is detected using a sex-specificchromosome. For example, alleles at a heterozygous X chromosome testlocus can be detected, with the presence of one allele in at least onesubsample and the presence of both alleles in at least one othersubsample indicating the presence of mosaic aneuploidy (e.g., mosaicTurner syndrome). In another example, the presence of alleles at ahomozygous X chromosome test locus can be detected, with a 1:1 X:Ychromosome intensity ratio in at least one subsample and a 2:1 X:Ychromosome intensity ratio in at least one other subsample indicatingthe presence of a mosaic aneuploidy (e.g., mosaic Klinefelter syndrome).

Screening or genotyping aliquots or subsamples on a cell-by-cell basisalso allows for the detection of dizygotic twins (i.e., non-identicaltwins). For example, SNP genotyping can be performed on subsamplescontaining a fetal allele, with the presence of at least two subsampleswith different SNP genotypes indicating the presence of dizygotic twins.In some embodiments, the number of SNPs screened or genotyped to detectdizygotic twins is about 1 to about 20 SNPs. In some embodiments, thenumber of SNPs screened or genotyped is, is about, is at least, is atleast about, is not more than, is not more than about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 SNPs, or a rangedefined by any two of the preceding values. In a preferred embodiment,the number of SNPs screened or genotyped is about 3 to about 4 SNPs. Theprobability of detecting the presence of dizygotic twins using 3 SNPs isabout 1−(0.5)(0.5)(0.5)=87.5%, while the probability of detecting thepresence of dizygotic twins using 4 SNPs is about1−(0.5)(0.5)(0.5)(0.5)=93.75%, respectively.

In some embodiments, aliquots containing fetal alleles from dizygotictwins can be pooled. For example, aliquots comprising cells from a firsttwin can be pooled independently of aliquots comprising cells from asecond twin. In some embodiments, pooled aliquots from a first andsecond twin can be independently placed on one or more arrays andassessed for genetic or chromosomal variation as described herein.

Techniques

Even if fetal DNA or RNA is present in a minor fraction of a samplecomprising maternal and fetal genetic material, it is still possible todetect fetal SNP alleles using standard SNP detection formats, such asTAQMAN® PCR. However, to detect minor alleles, it may be necessary tooptimize fluorescence detection to prevent maternal signal overlap fromobscuring a fetal-specific signal. In some embodiments, optimalfluorescence dyes for probe labeling are selected to minimize overlap.For example, dyes separated across the standard fluorescence spectrum(400-700 nm) with less than 10% overlap between emissions can beselected. In this way, even minor signals (less than 10% signalintensity) from fetal alleles are not obscured by maternal signal (90%signal intensity). In some embodiments, optimal fluorescence filters arechosen to minimize overlap in emission detection and custom fluorescencedetection hardware and software are chosen to minimize signal crosstalk.In some embodiments, digital PCR is used to optimize signal tobackground ratios.

Other potential tools for measuring allelic intensity include S-curvefitting and other statistical analyses known in the art. In someembodiments, Ct shift is measured to detect a genetic or chromosomalvariation. For example, encapsulation of single cells for detection offetal SNPs with TAQMAN® chemistry can allow for simultaneous detectionof abnormal copy number for the SNP detected, and therefore the copynumber of the corresponding chromosome. If a SNP resides on chromosome21, the SNP abundance will correlate with a Ct value, and therefore acopy number for Chromosome 21. If a reaction contains a single cell,then the copy number of a chromosome in that cell can be detected by Ctshift. In this example, the use of the abundance of normal maternal cellCt values establishes the Ct for a normal copy number of two chromosomesper cell and a Ct shift to an earlier cycle would indicate the presenceof a copy number variation.

As described herein, sequencing methods can be used to screen for fetalalleles and/or to determine the presence of a genetic or chromosomalvariation. In some embodiments, shotgun sequencing may be used as analternative to CGH arrays to detect copy number variations (e.g.,resulting from a genetic variation) as described, for example, in Xieand Tammi, BMC Bioinformatics 2009, 10(80). In some embodiments, wholegenome sequencing may be performed.

SNP genotyping can also be performed using any method known in the art,including qPCR and TAQMAN® methods. A variety of SNP chemistries andplatforms are available from manufacturers such as Life Technologies(TAQMAN®) (Carlsbad, Calif.), Illumina (GOLDENGATE®) (San Diego,Calif.), Millipore (AMPLIFLUOR®) (Billerica, Mass.), and DxS Ltd.(SCORPIONS™) (Manchester UK). Miniaturized formats are also availablefrom BioTrove (OPENARRAY™) (Woburn, Mass.) and Fluidigm (BIOMARK™)(South San Francisco, Calif.).

SNP Panels

A SNP panel can be used to identify target loci in a maternal geneticsample. Once these target loci are identified, they are used to identifythe presence of a non-maternal allele in a mixed sample. Becausegenotyping a maternal genetic sample to identify a target locus isexpensive and time consuming, a SNP panel is designed to include as fewSNPs as possible. However, the panel must still include enough SNPs toidentify a large enough set of target loci to allow for the detection ofa fetal allele in a mixed sample, with these SNPs being sufficientlyinformative to conserve the finite quantity of cells in a mixture offetal and maternal cells.

The size of a SNP panel is inversely related to the minor allelefrequencies of the SNPs in the panel. In some embodiments of theinvention, the goal is to identify about 1 to about 5 fetal cells from amixed sample of maternal and fetal cells. The number of SNPs that mustbe assessed to achieve this goal depends on the minor allele frequencyof SNPs that are assessed and the number of cells that are genotyped.The number of cells that must be assessed, in turn, depends on theextent of enrichment of the mixed sample for fetal cells.

In some embodiments, a SNP panel is therefore designed to minimize thenumber of tests necessary to identify loci which are homozygous (or,alternatively, heterozygous) in a maternal sample and heterozygous (or,alternatively, homozygous) in a mixed sample of maternal and fetalgenetic material. In some embodiments, a SNP panel is designed toidentify about 1 to about 20 homozygous maternal SNPs perchromosome-specific SNP panel. In some embodiments, the SNP panel isdesigned to detect a number of homozygous maternal SNPs perchromosome-specific panel that is, is about, is at least, is at leastabout, is not more than, is not more than about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 SNPs, or a rangedefined by any two of the preceding values. In a preferred embodiment, aSNP panel is designed to identify about 5 to about 10 homozygousmaternal SNPs per chromosome-specific SNP panel. More preferably, a SNPpanel is designed to identify about 7 homozygous maternal SNPs perchromosome-specific SNP panel. For example, as shown in Table 2, forSNPs in HWE where p²=0.25, a panel of 20 SNPs is needed to identify 10SNPs for which the maternal sample is homozygous.

Several chromosome-specific SNP panels, preferably comprising at leastone control chromosome-specific panel, can be combined to create a SNPpanel for genotyping maternal genetic material. Each chromosome-specificpanel is designed to generate a target set of loci for that chromosome.Preferably, a chromosome-specific SNP panel comprises about 5 to about100 unique SNPs. Preferably, the total number of SNPs in achromosome-specific panel is between about 5 and about 30 unique SNPs.More preferably, the total number of SNPs in a chromosome-specific panelis about 20 SNPs. In some embodiments, the total number of SNPs in achromosome-specific panel is, is about, is at least, is at least about,is not more than, is not more than about 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, or 100 SNPs, or a range defined by any two of the precedingvalues.

SNP panels may contain one or more chromosome-specific panels. Achromosome-specific SNP panel can comprise SNPs located on autosomalchromosomes, preferably SNPs located on chromosomes that are susceptibleto aneuploidy in clinical relevant syndromes. More preferably,chromosome-specific SNP panels comprise SNPs located on Chromosome 13,18, 21, X, and Y. A chromosome-specific panel can also be a controlchromosome-specific panel. Preferably, a control chromosome-specificpanel comprises SNPs located on a chromosome that is not susceptible toaneuploidy or where the aneuploidy is incompatible with viability, whichis typically the larger chromosomes that are designated by lower indices(e.g., chromosome 1, 2, or 3). Most preferably, a controlchromosome-specific panel comprises SNPs located on Chromosome 1, 2, or3. In addition, a chromosome-specific SNP panel can also comprise SNPslocated on sex-specific chromosomes. In some embodiments, a panel is notspecific for a particular chromosome. In some embodiments, the controlis not a chromosome-specific SNP panel. For example, primers can be usedto amplify a chromosome-specific region which will serve as a control.

In some embodiments, a SNP panel comprises more than onechromosome-specific panel, where the chromosome-specific panels are forSNPs on Chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, X, or Y. In some embodiments, the SNP panelcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, or 23 chromosome-specific panels, or a range defined byany two of the preceding values. In some embodiments, the total numberof SNPs in the panel is N*the number of SNPs on a chromosome-specificpanel, where N is the number of chromosome-specific panels in the SNPpanel.

Several factors can be considered in the selection of loci forgenotyping panels. For example, Hardy-Weinberg equilibrium (“HWE”) canbe assumed to calculate probabilities of SNP allele frequencies. In someembodiments, the probability of the alleles in the selected SNPs isgiven by the following equation: p²+2pq+q²=1. For example, SNPs with aheterozygosity of 0.5 can be selected. HWE is preferred, as SNPs thatare not in HWE are unlikely heritable SNPs, limiting their utility, ormay result from faulty genotyping chemistry.

SNPs with one allele favored in homozygosity across major knownhaplotypes can also be selected. In some embodiments, SNPs with ahomozygous maternal genotype (pp or qq) at less than 0.25 and anopposite homozygous genotype at more than 0.25 are selected.

In some embodiments, SNPs have a frequency in the range of about 30% toabout 50% for the minor allele as measured across all major populationgroups. In a preferred embodiment, SNPs have a frequency in the range ofabout 49% to about 50% for the minor allele. In some embodiments, SNPshave a frequency that is, is about, is at least, is at least about, isnot more than, is not more than about 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%for the minor allele, or a range defined by any two of the precedingvalues.

Embodiments of the Invention

One embodiment of the invention is a method of detecting a fetal allele,comprising: obtaining a sample comprising a mixture of maternal andfetal cells; enriching the sample for fetal cells; dividing the enrichedsample into subsamples; performing whole genome amplification on asubsample to provide an amplified genomic product; dividing theamplified genomic product into aliquots; identifying target loci atwhich the genetic material of the maternal cells is homozygous for aminor allele; selecting a test locus from the target loci; screening analiquot for a non-maternal allele (heterozygous genotype) at the testlocus, where the presence of the non-maternal allele (heterozygousgenotype) indicates the presence of a fetal allele in the amplifiedgenomic product; and identifying at least one fetal allele.

Another embodiment of the invention is a method of detecting a fetalgenetic variation, comprising: a method of identifying a fetal allele ina mixed sample of maternal and fetal cells, comprising: obtaining asample comprising a mixture of maternal and fetal cells; enriching thesample for fetal cells; dividing the enriched sample into subsamples,each subsample comprising not more than one cell; identifying a panel ofat least one locus at which the genetic material of the maternal cellsis homozygous for a minor allele; selecting a test locus from the targetloci; screening a subsample for a non-maternal allele (heterozygousgenotype) at a test locus selected from the panel, where the presence ofthe non-maternal allele (heterozygous genotype) indicates the presenceof a fetal allele in the subsample; and identifying at least one fetalallele; and determining a ratio of maternally- and paternally-inheritedalleles in a subsample comprising the fetal allele, where the ratio isanalyzed to determine the presence of a genetic variation.

Another embodiment of the invention is a method of detecting a fetalallele, comprising: obtaining a sample comprising a mixture of maternaland fetal cells; dividing the sample into subsamples; performingpreamplification on a subsample to provide an amplified product;dividing the amplified product into aliquots; selecting target loci anddetermining whether the genetic material of the maternal cells ishomozygous or heterozygous at a set of target loci to determine amaternal genotype; selecting a test locus from the target loci;screening an aliquot for a genotype differing from the maternal genotypeat the test locus, where the presence of a genotype differing from thematernal genotype indicates the presence of a fetal allele in theamplified product; and identifying at least one fetal allele.

Another embodiment of the invention is a method of detecting a fetalallele, comprising: obtaining a sample comprising a mixture of maternaland fetal cells; dividing the sample into subsamples; identifying apanel of at least one target locus and determining whether the geneticmaterial of the maternal cells is homozygous or heterozygous for atleast one target locus to determine a maternal genotype; selecting atest locus from at least one target locus; screening a subsample for agenotype differing from the maternal genotype at the test locus selectedfrom the panel, where the presence of a genotype differing from thematernal genotype indicates the presence of a fetal allele in thesubsample; and identifying at least one fetal allele.

Embodiments of the invention can and may comprise one or more of thefollowing: a method further comprising enriching the sample for thefetal cells; a method where the enriching comprises differentialexpansion of the fetal cells over the maternal cells; a method furthercomprising analyzing the amplified product identified as containing thefetal allele to detect a fetal genetic variation; a method where thefetal genetic variation is selected from the group consisting of ananeuploidy, a microdeletion, a microduplication, and a mutation or othergenetic variation; a method further comprising analyzing the amplifiedproduct identified as containing the fetal allele to detect the presenceof mosaicism; a method further comprising analyzing the amplifiedproduct identified as containing the fetal allele to detect the presenceof dizygotic twins; a method where the preamplification comprises wholegenome amplification or whole transcriptome amplification; a methodwhere the target loci are homozygous for the maternal genotype; a methodwhere the target loci are heterozygous for the maternal genotype; amethod where the target loci comprise a mixture of homozygous andheterozygous loci for the maternal genotype; a method where theamplified product comprises genomic DNA; a method where the amplifiedproduct comprises complementary DNA; a method where determining amaternal genotype comprises using a panel of SNPs to genotype a sampleof maternal genetic material from the same individual that is the sourceof the maternal cells; a method where the source of the maternal geneticmaterial is selected from the group consisting of blood, serum, plasma,urine, a cervical swab, or a buccal swab; a method where the source ofthe maternal genetic material is selected from the group consisting ofblood or a buccal swab; a method where the selection of the test locuscomprises screening or genotyping a sample of maternal and fetalcell-free nucleic acids for a genotype differing from the maternalgenotype at a target locus, and selecting as the test locus a targetlocus with a non-maternal genotype in the sample of cell-free nucleicacids; a method where the non-maternal genotype is homozygous; a methodwhere the non-maternal genotype is heterozygous; a method where thesource of the maternal and fetal cell-free nucleic acid sample is blood,serum, plasma, urine, or a cervical swab from a pregnant woman; a methodcomprising selecting and screening more than one test locus in a singlealiquot; a method comprising selecting and screening more than one testlocus in more than one aliquot; a method comprising selecting andscreening one test locus in a single aliquot; a method comprisingselecting and screening one test locus in more than one aliquot; amethod where each subsample comprises not more than one cell; a methodfurther comprising pooling groups of two or more aliquots prior toscreening; a method where pooling comprises the use of an indexed systemof rows and columns of wells comprising the aliquots; a method where thenumber of rows and columns are independently between about 2 and about1000; a method where the number of rows and columns are independentlybetween about 8 and about 100; a method where the number of rows andcolumns are independently between about 16 and about 24; a method whereat least one target locus is homozygous for the maternal genotype; amethod where at least one target locus is heterozygous for the maternalgenotype; a method where the panel comprises a mixture of homozygous andheterozygous target loci; a method further comprising determining asignal intensity in a subsample comprising the fetal allele, where thesignal intensity is analyzed to determine the presence of a geneticvariation; a method further comprising determining an overrepresentationor underrepresentation of chromosomal sequences in a subsamplecomprising the fetal allele compared to a subsample comprising thematernal allele, wherein the overrepresentation or underrepresentationis used to determine the presence of a chromosomal aneuploidy,microdeletion, or microduplication; a method further comprisingdetermining a signal intensity in a subsample comprising the fetalallele, wherein the signal intensity is analyzed to determine thepresence of mosaicism; a method further comprising determining a copynumber of alleles in a subsample comprising the fetal allele, where thecopy number of alleles is analyzed to determine the presence ofmosaicism; a method further comprising determining a copy number ofalleles in a subsample comprising the fetal allele, where the copynumber of alleles is analyzed to determine the presence of a geneticvariation; a method where determining a maternal genotype comprisesusing a panel of SNPs to genotype a sample of maternal genetic materialfrom the same individual that is the source of the maternal cells; amethod where the target locus is homozygous and the test locus isheterozygous; a method where the target locus is heterozygous and thetest locus is homozygous; a method where the panel comprises a mixtureof homozygous and heterozygous target loci; a method where the panel ofSNPs is the panel of SNPs as described herein; and a method where thepanel is a panel of at least five loci.

Another embodiment of the invention is a method of detecting aninformative paternal allele, comprising: obtaining a sample comprising amixture of maternal and fetal cells; optionally enriching the sample forthe fetal cells; dividing the sample or enriched sample into subsamples;optionally dividing the amplified product into aliquots; optionallyselecting a set of target loci; determining whether the genetic materialof the maternal cells is homozygous or heterozygous at a set of targetloci to determine a maternal genotype; optionally selecting a test locusfrom the set of target loci, where selection optionally comprisesscreening a sample of mixed maternal and fetal nucleic acids for agenotype differing from the maternal genotype at a target locus, andselecting as the test locus a target locus with a non-maternal genotypein the sample of mixed maternal and fetal nucleic acids; screening orgenotyping an aliquot or subsample for the presence of an informativepaternal allele, where screening optionally comprises screening thealiquot for a genotype differing from the maternal genotype at at leastone target or test locus, and where the presence of the genotypediffering from the maternal genotype indicates the presence of aninformative paternal allele in the aliquot; and identifying the presenceof at least one informative paternal allele in at least one aliquot. Insome embodiments, the method further comprises: optionally collecting aportion of the aliquot identified as containing the informative paternalallele; and analyzing an aliquot or a collected portion of the amplifiedproduct identified as containing the informative paternal allele todetect a genetic variation.

Some embodiments of the invention comprise: a SNP panel where thechromosome-specific panel is directed to a chromosome subject toaneuploidy selected from the group consisting of Chromosomes 13, 18, and21, X, and Y; and a SNP panel further comprising a controlchromosome-specific panel directed to a control chromosome selected fromthe group consisting of Chromosomes 1, 2, and 3.

The term screening as used in at least one of the priority documentscited above encompasses the methods of screening and genotypingdescribed herein. In addition, the terms homozygous and heterozygousalleles as used in at least one of the priority documents cited aboveare sometimes referred to herein as heterozygous or homozygous locus orloci, or heterozygous or homozygous genotypes. Further, the termpreamplification as used in at least one of the priority documents citedabove is referred to herein as subsample amplification.

It will be understood by those of skill in the art that numerous andvarious modifications can be made without departing from the spirit ofthe present invention. All the references referred to herein areincorporated by reference in their entirety for the subject matterdiscussed. The following examples are included for illustrative purposesonly and are not intended to limit the scope of the invention.

EXAMPLES Example 1

The following example of an embodiment of the invention involvesPCR-based detection of a fetal sample, followed by WGA. Cells,optionally an enriched mixture of maternal and fetal cells, aresuspended in a buffer such as PBS and distributed into a second bufferwith appropriate chemistry for cell lysis and PCR. The cells in a PCRcompatible buffer are then partitioned into individual reactions, forexample by the introduction of oil plugs to create oil separation ofindividual cells in lysis and PCR buffer. This method preferably uses achemistry that fulfills a single-step cell lysis, and amplification oftarget nucleic acids without addition of reagents followingencapsulation. This method also preferably does not involve DNApurification prior to DNA amplification. Each nominal single-cellreaction is then subjected to heating and cooling thermal cycling withfetal specific fluorescent probe detection either during the thermalcycling or at the end of the final cycle and detection of allcompartments that contain fetal cells (i.e., one or more). In thepreferred case, one cell per reaction is obtained, but reactionsconsisting of one fetal cell and a few to many maternal cells will stillcorrectly identify the presence or absence of a fetal cell. So long asthe subsequent genomic analysis tolerates the equivalent contamination(i.e., one fetal cell and two maternal cells implies 33% pure fetalDNA), then more than one cell per reaction is acceptable. Each positivecompartment is optionally pooled into a single shared vessel. Thispooled, fetal-specific reaction is subjected to DNA clean-up procedures,such as the removal of RNA and proteins. Following the optional cleanup,the nucleic acids are subject to WGA using any compatible chemistry. Themethods for WGA include any of the following: Degenerate oligonucleotideprimed polymerase chain reaction (“DOP-PCR”), Ligation mediated PCR(“LM-PCR”), Strand Displacement amplification (“SDA”) with φ-29polymerase, or other combination protocols (Rubicon Genomics, or NuGen).Following WGA, the resulting amplified genomic DNA is used for geneticanalysis, for example, comparative genomic hybridization (“CGH”) orcomplete genome sequencing. There are many methods that are used forCGH, including BAC arrays or oligo arrays. There are also a variety ofmethods for sequencing including standard capillary based Sangersequencing and current “next-generation” high throughput sequencingplatforms, including those offered by Solexa/Illumina (San Diego,Calif.), Complete Genomics (Mountain View, Calif.), Roche/454 (Branford,Conn.), and Life Technologies (SOLiD™) (Carlsbad, Calif.).

Example 2

The following example of an embodiment of the invention for detection offetal DNA from cells includes isolation of cells into discrete reactionchambers followed by WGA, then separation of each reaction into multiplealiquots for PCR fetal allele detection, and then optionally poolingconjugate aliquots of fetal positive reactions for genetic analysis,e.g., CGH or sequencing. Cells, optionally an enriched mixture of fetaland maternal cells, are distributed into isolated reaction chamberscontaining appropriate buffers and reagents for cell lysis and WGA. Thiscould be in alternating aqueous and oil phases in capillaries or in aplate format with access to allow multistep reagent addition. In a plateformat, cells are first resuspended as about 100,000 cells in 200 μl,and placed into a well of a 96-well plate. Then using standardmicropipetting techniques, such as those offered by TECAN (Männedorf,Switzerland), LabCyte (Sunnyvale, Calif.) or others, 10 nL of cellsuspension containing an average of 1, 2, 3, 4, or 5 cells aretransferred to a high density plate containing, for example, 1,536 or3,456 wells. Standard WGA chemistries are used. For example, to each 10nL of cell volume, 90 nL of reagent for WGA is added, the plate issealed by oil to prevent evaporation, and WGA chemistry is allowed toproceed. Following WGA, up to 25 nL, containing many copies of amplifiedgenomic DNA from all cells in the reaction, are transferred to a secondhigh-density plate. Then an aliquot of up to 100 nL of PCR reagentcontaining primers and probes to identify fetal DNA containing reactionsis screened using a target locus from a SNP panel as disclosed herein.Reactions which are positive for fetal DNA are used, optionally afterpooling, for downstream genomic analysis, including CGH or sequencing.The reactions are subjected to clean up procedures prior to CGHhybridization or sequencing.

Example 3

In the following example of an embodiment of the invention, SNPs locatedon Chromosome 21 are selected for a panel. Hardy-Weinberg equilibrium(“HWE”) is assumed to calculate probabilities of allele frequencies. Oneallele is favored in homozygosity across the major known haplotypes. Thematernal genotype is homozygous (pp or qq) at less than 0.25 and theopposite homozygous genotype is at more than 0.25, and the probabilitiesof the alleles is given by the following equation: p²+2pq+q²=1. The SNPsare inherited in a Mendelian fashion.

Example 4

In the following example of an embodiment of the invention, a proposedpanel involves selecting SNPs at HWE, where the ratios of p² and q² are0.25, and heterozygosity is 0.5. With these ratios, finding homozygousmaternal SNPs requires statistically only 20 SNPs to find 10 SNPs wherethe maternal sample is homozygous (20*(0.25+0.25)). For any givenhomozygous SNP, the population including the fetus would have aprobability of being heterozygous of 0.5. For the first maternalhomozygous SNP, the probability of fetal heterozygosity is 0.5, for atotal probability of 0.5. For the first and second maternal homozygousSNP, the combined probability is 1−(0.5*0.5)=0.75. The combinedprobabilities of seven SNPs is 0.992.

Example 5

The following example is an embodiment of the invention. A pregnantfemale presents at 14 weeks gestational age. To detect whether the fetushas trisomy 21, a physician obtains a 30 mL blood sample and epithelialcells from the pregnant female via venipuncture and buccal swab,respectively.

A panel of SNPs is designed. Twenty SNPs each from Chromosomes 1 and 21(i.e., 40 target loci) are selected for the panel, with each SNP havinga minor allele frequency of 49% across all major populations. MaternalDNA is extracted from the buccal swab and genotyped using the SNP panel.Twenty SNPs from the panel generate a homozygous genotype (i.e., targetloci) and are selected for further genotyping in the mixture of maternaland fetal cells from the blood sample.

The blood sample is enriched to 1:10,000 fetal:maternal cells bysubjecting the sample to FICOLL™ ([location]), followed by Miltenyimagnetic beads (Gladbach, Germany). The enriched blood sample issuspended in a solution containing lysis buffer and PCR reagents. Thesample is then combined with oil plugs and introduced to a microfluidicdevice such that the sample is divided into subsamples that eachcomprise not more than one cell. Once inside the microfluidic device,the subsamples undergo a single-step cell lysis, followed by PCRamplification as described below.

A calculation is performed to determine that with a concentration of1:10,000 fetal:maternal cells, 50,000-cells must be screened to detectabout 5 heterozygous loci for a SNP with a minor allele frequency of49%.

A first set of 50,000 subsamples (and therefore 50,000 cells) isscreened subsample-by-subsample (and therefore cell-by-cell) for a firstSNP on each of Chromosomes 1 and 21 (i.e., a total of two loci) usingmultiplex qPCR with the TAQMAN® system. No heterozygous genotypes areidentified for either of the SNPs on Chromosome 1 or 21. A second set of50,000 subsamples is then screened subsample-by-subsample for a secondSNP on each of Chromosomes 1 and 21. The SNP on Chromosome 1 isheterozygous (indicating the presence of a fetal allele), while the SNPon Chromosome 21 is homozygous. A third set of 50,000 subsamples is thenscreened subsample-by-subsample for a third SNP on each of Chromosome 1and 21. This time, the SNP on Chromosome 21 is heterozygous and nofurther genotyping is performed.

The ratio of alleles from the third SNP on Chromosome 21 is thenanalyzed to detect the presence of aneuploidy. Because the allelicintensity of the maternally-derived allele (C) compared to thepaternally-derived allele (G) is 2:1 (i.e., CCG), trisomy 21 isdetected.

Example 6

The following example is an embodiment of the invention. Fetal and adultcells were grown in the presence of different factors to assess theireffect on differential expansion of fetal cells. The factors were StemCell Factor at 50 ng/mL, IL-3 at 5 ng/nL, IL-6 at 5 ng/mL, EPO at 1.5LVmL, TPO at 100 ng/mL, and Flt-3 at 50 ng/mL. The cells were CD34+positive cells purified from adult mobilized donor peripheral blood andCD34+ positive cells purified from fetal liver tissue purchased fromCambrex (Walkersville, Md.). Cells were plated at 10,000 cells per mLinto 24-well tissue culture plates. Cells were incubated in HPGM mediumwith 50 units/mL of penicillin, 50 μg/mL streptomycin sulfate, and thecytokine combinations above for 6 days at 37° C. and 5% CO₂ in ahumidified chamber. After six days, an aliquot of cells was countedmanually with a hemacytometer and the total cell numbers were calculatedusing a standard formula. An additional aliquot was used to assay totalATP levels (linear correlation with total cell numbers) using aVIALIGHT® assay kit (Cambrex, Walkersville, Md.).

In every case measured, the fetal cells expanded more than adult cells(e.g., compare the fourth column to the seventh column from Table 4).The final column of Table 4 shows the ratio of fetal cells to adultcells for some grow conditions. In every case where the ratio wasmeasured, fetal cells were more numerous after expansion.

Example 7

The following example of an embodiment of the invention for detection offetal DNA from cells includes isolation of cells into discrete reactionchambers followed by WGA, and direct detection of genetic variation.Cells, optionally an enriched mixture of fetal and maternal cells, aredistributed into isolated reaction chambers containing appropriatebuffers and reagents for cell lysis and WGA. In a plate format, cellsare first resuspended as about 100,000 cells in 200 μl, and placed intoa well of a 96-well plate. Then using standard micropipettingtechniques, such as those offered by TECAN (Männedorf, Switzerland),LabCyte (Sunnyvale, Calif.), or others, 10 nL of cell suspensioncontaining an average of 1, 2, 3, 4, or 5 cells are transferred to ahigh density plate containing, for example, 1,536 or 3,456 wells.Standard WGA chemistries are used. For example, to each 10 nL of cellvolume, 90 nL of reagent for WGA is added, the plate is sealed by oil toprevent evaporation, and WGA chemistry is allowed to proceed. FollowingWGA, PCR reagent containing primers and probes to identify fetalDNA-containing reactions is added to amplify a target locus from a SNPpanel as disclosed herein. Reactions which are positive for fetal DNAare directly detected using capture probes in the reaction chambers.Following target hybridization, wells are washed to remove non-targetDNA and non-reacted reagents. The specifically hybridized products arethen detected using standard chemistries, such as molecular beacons. Theratio of fetal and maternal sequences can then be used to determinegenetic variation.

Example 8

The following example is an embodiment of the invention. 40 ml of bloodis collected from a pregnant female and combined with a standardanticoagulant (e.g., EDTA), then shipped to a processing location. 12 mlof plasma is removed from the blood and cell-free DNA is extracted. Analiquot of whole blood or PBMCs is removed for maternal genotyping.

Gradient enrichment is performed for red blood cell removal, includingred blood cell lysis. Magnetic activated cell sorting (MACS) is used todeplete maternal cells to a yield of less than about 500,000 totalcells. Cell sorting is used to enrich the fetal fraction for a totalyield of about 10,000 cells. The cells are divided to λ=about 1 cell perreaction in a suitable volume. Cells are lysed directly and a PCRcompatible solution is created, possibly with the addition of aneutralization solution. WGA reagents are added to each reaction for atotal reaction volume of 100 nl. WGA is allowed to proceed for a totalof between about 10 and 10,000 total genomic equivalents of DNA (where areaction initially contained one genomic equivalent). The WGA reactionis stopped, and all reactions are split into at least oneposition-indexed aliquot. PCR is performed on all aliquots to detect anon-maternal allele (e.g., by comparing to a genotype obtained using thealiquot of whole blood or PBMCs). The original location of the WGAmaterial containing non-maternal alleles is identified, and the volumeat these locations is recovered. These volumes are then pooled for afinal WGA to generate a quantity of DNA suitable for aCGH (e.g., about0.5-1 ug). aCGH is then performed to detect CNVs.

TABLE 2 Number of Possible Number Homozygous Maternal of SNPs with SNPsp² 2pq q² Cumulative Testing 2 0.25 0.5 0.25 1 4 0.25 0.5 0.25 2 6 0.250.5 0.25 3 8 0.25 0.5 0.25 4 10 0.25 0.5 0.25 5 12 0.25 0.5 0.25 6 140.25 0.5 0.25 7 16 0.25 0.5 0.25 8 18 0.25 0.5 0.25 9 20 0.25 0.5 0.2510

TABLE 3 Cumulative probability of finding at least one heterozygousfetal allele, if the selected maternal p² 2pq q² SNPs are p² type SNP1 0.25 0.5 0.25  50.00% SNP2  0.25 0.5 0.25  75.00% SNP3  0.25 0.5 0.25 87.50% SNP4  0.25 0.5 0.25  93.75% SNP5  0.25 0.5 0.25  96.88% SNP6 0.25 0.5 0.25  98.44% SNP7  0.25 0.5 0.25  99.22% SNP8  0.25 0.5 0.25 99.61% SNP9  0.25 0.5 0.25  99.80% SNP10 0.25 0.5 0.25  99.90% SNP110.25 0.5 0.25  99.95% SNP12 0.25 0.5 0.25  99.98% SNP13 0.25 0.5 0.25 99.99% SNP14 0.25 0.5 0.25  99.99% SNP15 0.25 0.5 0.25 100.00% SNP160.25 0.5 0.25 100.00% SNP17 0.25 0.5 0.25 100.00% SNP18 0.25 0.5 0.25100.00% SNP19 0.25 0.5 0.25 100.00% SNP20 0.25 0.5 0.25 100.00%

TABLE 4 Total Fold Total Fold Differential Differential CytokineATP/well Fetal cells expansion ATP/well Adult cells expansion ATP countCombination (AU) per well (fetal) (AU) per well (adult) Fetal/AdultFetal/Adult O 77.2 188500 18.85 0.8 7500 0.75 93.6 25.1 OA 272.8 57750057.75 46.2 91200 9.12 5.9 6.3 OB 167.9 410400 41.04 8.9 23400 2.34 18.817.5 OC 657.9 1410000 141 100.7 127500 12.75 6.5 11.1 OD 267.0 65250065.25 18.3 37000 3.7 14.6 17.6 OE 213.5 663300 66.33 70.9 150000 15 3.04.4 OAB 280.2 57.5 4.9 OAC 668.3 282.6 2.4 AOD 457.9 95.0 4.8 OAE 517.1186.0 2.8 OBC 741.4 225.8 3.3 OBD 335.9 850500 85.05 38.7 123200 12.328.7 6.9 OBE 341.2 138.6 2.5 OCD 706.0 260.2 2.7 OCE 638.6 338.1 1.9 ODE480.5 157.6 3.0 OABC 691.0 323.9 2.1 OABD 389.8 92.8 4.2 OABE 281.3126.5 2.2 OACD 689.6 326.3 2.1 OACE 609.6 347.3 1.8 OADE 508.0 181.6 2.8OBCD 694.0 268.7 2.6 OBOE 543.4 228.4 2.4 OBDE 299.0 122.2 2.4 OCDE670.0 283.4 2.4 OABCD 568.6 259.4 2.2 OABCE 602.1 289.0 2.1 OABDE 317.4137.4 2.3 OACDE 472.0 165.0 2.9 OBCDE 441.0 1449000 144.9 215.1 29200029.2 2.1 5.0 OABCDE 571.3 1577000 157.7 259.8 440800 44.08 2.2 3.6 OC1000000 100 220000 22 4.5 OCD 1200000 120 350000 35 3.4 O 450000 4530000 3 15.0 OA 75000 7.5 50000 5 1.5 OB 550000 55 30000 3 18.3 OAB625000 62.5 100000 10 6.3 OE 900000 90 130000 13 6.9 ODE 1250000 125160000 16 7.8 O: Stem Cell Factor (50 ng/mL) A: IL-3 (5 ng/mL) B: IL-6(5 ng/mL) C: EPO (1.5 U/mL) D: TPO (100 ng/mL) E: Flt-3 (50 ng/mL)

What is claimed is:
 1. A method of obtaining and analyzing a fetalgenome from a mixture of maternal and fetal cells, comprising: a)obtaining a maternal blood sample comprising a mixture of maternal andfetal cells; b) enriching said sample from step a) for said fetal cells;c) dividing said enriched sample from step b) into at least about 100subsamples; d) performing whole genome amplification on each of the atleast about 100 subsamples from step c) to provide an amplified productfor each subsample, wherein each amplified product has an amplifiedgenome from an average of about one cell, and wherein at least 99% ofthe amplified genomes in said amplified product are not from a fetalcell; e) dividing each of said amplified product from step d) intoaliquots, wherein each aliquot contains at least one copy of saidamplified genome from said cell, and wherein at least 99% of theamplified genomes in said aliquots are not from a fetal cell; f)screening or genotyping an aliquot from step e) for the presence of afetal identifier, wherein said screening or genotyping comprises: i)determining a maternal genotype and a fetal genotype of at least 1SNP(s) per chromosome-specific SNP panel on at least 10chromosome-specific panels; ii) selecting as test loci from step i) atleast 1 SNP(s) per chromosome-specific panel on at least 10chromosome-specific panels where the maternal genotype is homozygous andthe fetal genotype is heterozygous; iii) screening or genotyping saidaliquot from step e) for a genotype differing from a maternal genotypeat at least one of said test loci from step ii) , wherein the presenceof said genotype differing from said maternal genotype indicates thepossible presence of an informative paternal allele in said aliquot andthus said corresponding subsample; iv) screening or genotyping analiquot from a subsample having an informative paternal allele from stepiii) for a genotype differing from a maternal genotype at at least 1test loci per chromosome-specific panel on at least 10chromosome-specific panels from step ii), wherein the presence of saidgenotype differing from said maternal genotype indicates the presence ofan informative paternal allele in said aliquot, g) identifying thepresence of a fetal cell in at least one subsample from step c) whosealiquot contains an informative paternal allele from step f) iv); and h)analyzing another aliquot from step e) from said at least one subsamplefrom step g) identified as containing a fetal cell to detect a geneticvariation in said genome of said fetal cell.
 2. The method of claim 1,wherein said genetic variation is selected from the group consisting ofa chromosomal rearrangement, a copy number variation, and apolymorphism.
 3. The method of claim 1, wherein said analyzing to detecta genetic variation comprises determining a ratio of maternally- andpaternally-inherited alleles, wherein said ratio is analyzed todetermine the presence of a genetic variation.
 4. The method of claim 1,wherein said analyzing to detect a genetic variation comprisesdetermining a copy number of alleles in said aliquot, wherein said copynumber of alleles is analyzed to determine the presence of a geneticvariation.
 5. The method of claim 1, wherein said maternal genotype andfetal genotype are determined from a mixture of maternal and fetalnucleic acids from a mixture of cell-free nucleic acids.
 6. The methodof claim 5, wherein the source of said maternal and fetal cell-freenucleic acid sample is blood, serum, plasma, urine, cervical swab,cervical lavage, uterine lavage, or culdocentesis from a pregnantfemale.
 7. The method of claim 1, further comprising pooling an aliquotfrom two or more subsamples prior to screening or genotyping an aliquotfor the presence of a fetal identifier, wherein said screening orgenotyping further comprises: individually screening or genotyping asecond aliquot from the subsamples whose pooled aliquots contained aninformative paternal allele.
 8. The method of claim 7, wherein saidpooling comprises the use of an indexed system of rows and columns ofwells comprising said aliquots.
 9. The method of claim 1, whereinaliquots from two or more subsamples identified as containing a fetalcell are pooled prior to said analyzing another aliquot from said atleast one subsample identified as containing fetal cell to detect agenetic variation in said fetal cell.
 10. The method of claim 7, whereinaliquots from two or more subsamples identified as containing a fetalcell are pooled prior to said analyzing another aliquot from said atleast one subsample identified as containing fetal cell to detect agenetic variation in said fetal cell.
 11. The method of claim 1, whereinthe loci used to determine the presence of an informative paternalallele is not the same loci used to detect a genetic variation.